JP2002034599A - Method for detecting mono base polymorphism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an analysis method for nucleic acid sequence, capable of readily detecting the kind of a base polymorphism in a specific nucleic acid sequence containing a mono base polymorphism contained in a specimen. SOLUTION: This method detects a mono base polymorphism on a sequence of a chromosome or its fragment. The method for detecting the kind of a base to be specified comprises reacting a reaction solution comprising a nucleic acid primer containing a base adjacent to a polymorphism base at the 3' end, any one of dideoxynucleotide corresponding to a polymorphism base having an identifiable characteristic or its mixture, a DNA polymerase and a composition necessary for activity expression of the DNA polymerase with a specimen nucleic acid and detecting the presence of incorporation of the dideoxynucleotide into the nucleic acid primer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for analyzing a nucleic acid sequence. More specifically, the present invention relates to a nucleic acid sequence analysis method capable of detecting a nucleotide polymorphism contained in a specific nucleic acid sequence. The nucleic acid sequence analysis method of the present invention is useful in genetic engineering, molecular biology and related industrial fields.

[0002]

BACKGROUND OF THE INVENTION In the present invention, a nucleotide polymorphism is a genotype having a sequence different from that of a wild type, and the polymorphism gene is important as a cause of inter-individual variation in the occurrence of side effects and treatment failure in drug metabolism. Plays a role. It is also known as a cause of individual differences such as basal metabolism known as a constitution. Moreover, they also serve as genetic markers for a number of diseases. Therefore, the elucidation of these mutations is clinically important, and routine phenotyping is particularly recommended for psychiatric patients and volunteers in clinical studies (Gram and Brsen, European Consen
sus Conference on Pharmacogenetics. Commission of
the European Communities, Luxembourg, 1990, pp87-9
6; Balant et al., Eur. J. Clin. Pharmacol. 36, 551-55.
4, 1989). Therefore, nucleic acid sequence analysis for the detection of each genotype following the identification of the causative polymorphic gene is desired.

[0003] As a conventional nucleic acid sequence analysis technique, there is, for example, a nucleic acid sequencing method (sequencing method). Nucleic acid sequencing can detect and identify nucleotide polymorphisms contained in nucleic acid sequences, but requires a great deal of labor to perform preparation of template nucleic acid, polymerase reaction, polyacrylamide gel electrophoresis, analysis of nucleic acid sequence, etc. Time is needed. In addition, although labor saving can be achieved by using a recent automatic sequencer, there is a problem that an expensive apparatus is required.

[0004] As another method, there is the Southern hybridization method (Laboratory Manual Engineering Enhancement Edition, edited by Masami Muramatsu, Maruzen Co., Ltd., pp. 70-75). According to this method, a region of DNA having a base sequence complementary to the labeled DNA probe can be identified.
That is, in the Southern hybridization method, a nucleic acid fragment is electrophoresed on a plate of an agarose gel or polyacrylamide gel, separated according to the size (length) of the fragment, denatured into a single strand, and the plate is treated with nitro. After attaching a membrane such as cellulose or nylon, transferring the nucleic acid fragment as it is in the electrophoresis pattern, fixing it, and forming a hybrid with a nucleotide polymorphism-specific DNA probe labeled with RI (radioactive isotope), complements the probe. Nucleic acid fragments on a typical membrane are detected by autoradiography or the like.

[0005] According to the Southern hybridization method, the electrophoresis position and molecular weight of the target DNA fragment can be determined. However, the operation such as electrophoresis and autoradiography requires a long time, and the analysis is performed quickly. If the probe is not strictly specific, a similar sequence for detecting the presence or absence of a nucleotide polymorphism is identified because the detection cannot be performed only by hybridization or not with the nucleotide polymorphism-specific DNA probe. There was a problem that it was difficult.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a nucleic acid sequence analysis method which can easily detect the type of nucleotide polymorphism in a specific nucleic acid sequence containing a nucleotide polymorphism contained in a sample. is there.

[0007]

Means for Solving the Problems The present inventors have diligently studied a method for identifying single nucleotide polymorphisms without performing complicated operations while retaining the advantages of the sequencing method (Sanger method). The invention has been completed.

That is, the present invention has the following configuration. (1) A method for detecting a single nucleotide polymorphism on the sequence of a chromosome or a fragment thereof, wherein the nucleic acid primer has a base adjacent to the polymorphic base at the 3 ′ end, and corresponds to a polymorphic base having distinguishable characteristics. Reacting a sample nucleic acid with a reaction solution containing any one of the dideoxynucleotides or a mixture thereof, a DNA polymerase and a composition necessary for expressing its activity,
A method for detecting the type of base to be identified by detecting the presence or absence of dideoxynucleotide incorporation into a nucleic acid primer. (2) The method according to (1), wherein the distinguishable feature of the incorporated dideoxynucleotide is a biotin group. (3) The method according to (1), wherein after the incorporation reaction, the nucleic acid primer is captured and detected by hybridizing with the capture probe. (4) The method according to (1), wherein the DNA polymerase is heat-resistant, and the sensitivity is improved by repeating nucleic acid denaturation and annealing / incorporation by a temperature cycle. (5) The ratio of nucleic acid primers is made non-uniform so that the strand on the side to be detected is preferentially amplified, and the sample nucleic acid used for detection is amplified in advance.
The method according to (1). (6) Compare the amplification primer that hybridizes to the same sample nucleic acid strand as the nucleic acid primer with the amplification primer that hybridizes to the opposite sample nucleic acid strand.
(5) The method according to (5), wherein the number is increased by 5 to 10 times. (7) After amplifying the sample nucleic acid used for detection in advance,
The method according to (1), wherein dephosphorylation of various deoxynucleotides that hinder detection of a nucleotide polymorphism is performed with phosphatase. (8) An oligonucleotide having a common sequence arbitrarily set in advance is bound to the 5 'end of each nucleic acid primer, and the nucleic acid primer is captured using a capture probe complementary to the oligonucleotide of the common sequence, followed by detection. The method according to (1), wherein

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The first feature of the present invention is to extend only a portion of a single nucleotide polymorphism to be specified based on the Sanger method which is a nucleic acid sequencing technique. Since only one base is extended, various nucleotides and the like used in the ordinary Sanger method are not required, and one kind of dideoxynucleotide (ddNTP) serving as a specific terminator is added. Further, a feature of the present invention is that a distinguishable feature is added to the specific terminator. This makes it possible to clearly identify the presence or absence of ddNTP incorporation. In addition, if a plurality of mutually distinguishable features are used, it goes without saying that a plurality of assays can be performed in one reaction by adding each of them to different ddNTPs.

A known label can be used as the distinguishable feature to be added to ddNTP. In the present invention, biotin is mainly used, because the flexibility of being able to add various labels in a subsequent step by binding to avidin was advantageous at the research stage. Obviously, other identifiable features could be used.

[0011] Distinguishing features include, for example, enzymes,
Labels such as biotin, a fluorescent substance, a hapten, an antigen, an antibody, a radioactive substance, and a luminophore are exemplified, and the detection can be performed by detecting the distinguishable characteristic after the addition reaction of ddNTP. Alternatively, the reverse primer may be labeled.

Examples of the enzyme include alkaline phosphatase, peroxidase and the like.

As a fluorescent substance, FITC, 6-FA
M, HEX, TET, TAMRA, Texas Red, C
y3, Cy5 and the like.

Haptens include biotin, digoxigenin and the like.

[0015] Examples of the radioactive substance include ³² P and ³⁵ S.

Examples of the luminophore include ruthenium.

The label may be bound to any position of ddNTP without affecting the extension reaction of the primer.

In a preferred embodiment, the nucleic acid primer incorporating ddNTP is captured and detected by hybridization. Generally, sequencing by the Sanger method is detected by electrophoretic analysis on a polyacrylamide gel, but this requires considerable effort and is expensive. In the present invention,
As an alternative detection method, a method is described in which a nucleic acid primer is captured by reverse hybridization using a capture probe, and a label attached to ddNTP is detected after washing. The probe used for hybridization may have an antisense sequence of a nucleic acid primer.However, the present inventors have further improved this and previously bonded an oligonucleotide having a common sequence to the 5 ′ end of the nucleic acid primer. And a method of capturing with a common probe is also presented. This eliminates the need to synthesize a probe for each item, reduces the cost due to mass production of probes, eliminates the need to set hybridization conditions for each item, and improves development efficiency. Specifically, in Example 2, a dA 20mer was attached to the 5 ′ end of the nucleic acid primer, and dT
Captured with a 20-mer probe.

In a further preferred embodiment, a heat-resistant DNA polymerase is used as an enzyme for incorporating ddNTP into a nucleic acid primer. Thereby, the incorporation efficiency can be improved by repeating the steps of denaturation, annealing, and extension (incorporation) multiple times in a temperature cycle.
The present invention has no 3 ′ → 5 ′ exonuclease activity
Taq DNA polymerase and Tth DNA polymerase are suitable.

The gene containing a single nucleotide polymorphism site, for example, a chromosome or a fragment thereof, contained in the sample nucleic acid is not particularly limited as long as it is a target nucleic acid containing a nucleotide polymorphism site that carries information on the target gene. Examples of the target nucleic acid, Alu sequence,
Examples include exons, introns, and promoters of the gene encoding the protein. More specifically, examples include genes related to various diseases including genetic diseases, drug metabolism, and lifestyle-related diseases (hypertension, diabetes, etc.). For example, ACE (Angiotensin I Converting Enzyme) gene is mentioned as hypertension.

In the present invention, a chromosome or a fragment thereof may be simply referred to as a nucleic acid. The term “mutated nucleic acid” refers to a wild-type nucleic acid in which one nucleotide is point-mutated and replaced by another nucleotide, or a nucleic acid containing one base inserted or deleted in a wild-type nucleic acid. Which nucleotide is mutated has been elucidated. It has been elucidated that the constitution and the like are different depending on such nucleotide polymorphisms, and the method of the present invention is a method for examining whether or not a nucleic acid in a sample has such an expected mutation. is there.

The length of the hybridizing portion of the nucleic acid primer, the detection primer and the capture primer in the present invention is usually 13 to 35 bases, preferably 16 to 35 bases.
30 bases.

The present inventors have also presented a method for preparing a sample used in the above reaction. In theory, genomic DNA
Although it is possible to detect directly from nucleic acids in specimens, it is difficult in practice to secure a sample that can obtain a sufficient signal.
Amplification of only a specific sequence is carried out by a nucleic acid amplification technique such as a lymerase chain reaction (Japanese Patent Publication Nos. 4-67960 and 4-67957). The present inventors have also amplified the sample nucleic acid by PCR, but the signal was not always high despite sufficient amplification. Therefore, as a result of diligent studies, it was found that the nucleic acid amplified by PCR was double-stranded, which inhibited the binding of the nucleic acid primer for detection. Furthermore, the number of strands on the side to be annealed can be increased in a single-stranded state by increasing the number of primers on the side to be annealed with the detection primer extremely (5 to 10 times) as compared with the primer for amplification on the opposite side. It was possible, and it was found that the signal was improved.

In addition, deoxynucleotides (dNTPs, which are usually represented by plural forms since they are inserted in four types) which are added when performing nucleic acid amplification in advance become an obstacle when detecting single nucleotide polymorphisms. In fact, one mole of ddNTP added during detection in moles
Remaining 1/0 dNTPs will increase the signal of the negative sample and render it unspecific. dNTP
Is easier to be incorporated than ddNTP. Therefore, it is necessary to remove dNTPs almost completely after amplification, and purification has mainly been performed using a column. In recent years, a spin column method using a microcentrifuge has been developed, and the operation has become quite simple, but still requires complicated operations such as dispensing and centrifugation of the reaction solution, and there is concern about contamination of PCR products. . The cost is more than 200 yen per sample.
As a result of intensive studies, the present inventors have found a method for inactivating dNTPs by treatment with a phosphatase (phosphatase). Specifically, 2 units of alkaline phosphatase are added per 5 microliters of the PCR product, and the mixture is incubated at 37 ° C. for 15 minutes. If used in the above SNP detection reaction as it is, phosphatase will degrade to the required ddNTP,
Heat at 0 ° C to 100 ° C for 15 minutes to inactivate phosphatase. It was confirmed that this reaction solution could be used for the SNP detection reaction as it was. Heating at 37 ° C and 80 ° C can use a thermal cycler for PCR, so the only additional step is to add phosphatase, which is easy to automate. Since the phosphatase to be used must be completely inactivated by a heating operation after the treatment, it is preferable that the phosphatase not have a very high heat stability, and bovine small intestine alkaline phosphatase (CIP) or the like is preferable. The cost of CIP is low and can be implemented for less than 40 yen per sample. This technology is
In addition to the SNP detection method, it has versatility applicable to many inspection technologies.

[0025]

The effects of the present invention will be further clarified by exemplifying embodiments of the present invention. However, this does not limit the effects of the present invention. Embodiment 1 FIG. Single nucleotide polymorphism detection of human ACE gene by combining PCR and the present invention SNP present at the 240th base from the transcription start point of human ACE (Angiotensin-I converting enzyme) gene (ACE240 T / A)
Is detected using the present invention. (1) Preparation of Required Oligonucleotides Oligonucleotide (forward primer for amplification of ACE gene) having the sequence shown in SEQ ID NO: 1
Oligonucleotide having the sequence shown in Sequence Listing / SEQ ID NO: 2 (reverse primer for ACE gene amplification), oligonucleotide having the sequence shown in Sequence Listing / SEQ ID NO: 3 (ACE240SNP detection primer), and Sequence Listing / SEQ ID NO: 4 An oligonucleotide having the sequence shown (ACE2
Oligonucleotide for 40SNP capture probe) was synthesized. The oligonucleotide for the ACE240SNP capture probe (SEQ ID NO: 4) has an amino linker arm at the 5 'end.

The oligonucleotides of SEQ ID NOs: 3 and 4 are completely complementary. (2) Binding of oligonucleotide for capture probe to microtiter plate The oligonucleotide for capture probe synthesized in (1) above was bound to the inner surface of the microtiter plate via its linker arm. Linker oligonucleotide is 50mM borate buffer (pH10) 100mM
Dilute to a concentration of 0.05 pmol / μL in a MgCl ₂ solution, dispense 100 μL each into a microtiter plate (MicroFLUOR B, Dynatech), and leave at room temperature for about 15 hours to convert the linker oligonucleotide into a microtiter plate. Bond to the inner surface of the plate. After that, 0.1pmol dNTP
Replace with 0.5% PVP (polyvinyl pyrrolidone) 5 × SSC,
Blocking for suppressing non-specific reaction was performed at room temperature for about 2 hours. Finally, it was washed with 1 × SSC and dried. (3) Amplification by PCR of a region containing a single nucleotide polymorphism to be specified Oligonucleotide (forward primer for amplification of ACE gene) having the sequence shown in SEQ ID NO: 1 and the sequence shown in SEQ ID NO: 2 Of the ACE gene from a human genomic DNA sample extracted from blood with the nucleic acid extraction kit “QIAamp” (Qiagen) using an oligonucleotide (reverse primer for ACE gene amplification)
Amplified by PCR. In these DNA samples, the base of ACE240 has been specified in advance by a normal sequencing method,
There are three types, TT / TA / AA. KOD DNA poly for PCR
Using merase (Toyobo), 35 cycles of amplification reaction were performed under the conditions specified by the manufacturer. However, since the purpose of this PCR is simply to amplify a specific region of the gene,
There are no particular restrictions on the method of PCR or the type of enzyme. In addition, PCR
Sometimes, a reverse primer was added 10 times the standard method to preferentially amplify the strand on the detected side (= PC in Table 1).
R described as "asymmetric"). Then, "QIAquic
k "column (Qiagen) purified (= described as pre-treated" column "in Table 1), or bovine small intestine alkaline phosphatase (Toyobo) treated at 37 ° C for 15 minutes and then treated at 80 ° C for 15 minutes ( = Described in Table 1 as "pretreatment" enzyme). (PCR composition (Regular method)) Total solution volume 25μL Forward primer 5pmol Reverse primer 5pmol (50pmol) × 10 buffer 2.5μL 2mM dNTP 2.5μL KOD DNA polymerase 1U Extracted DNA solution 100ng (PCR conditions) 94 ℃ ・ 5min 94 ℃ ・1 minute, 65 ° C for 2 minutes, 68 ° C for 1 minute (35 cycles) 68 ° C for 2 minutes (4) Specific ddNTP incorporation reaction ACE240SNP detection primer was added to the PCR product prepared in (3).
A specific biotinylated ddNTP (B-ddNTP), DNA polymerase, etc. are added, and ddNTP is incorporated. This time the type of SNP
B-ddTTP (also possible with ddUTP) or B-dd
ATP was used. In addition, a modified Tth DNA polymerase “Delt
a-Tth DNA polymerase "(Toyobo) was used. B-ddTT
P is used in large quantities because it has lower uptake efficiency than B-ddATP. (DdNTP uptake reaction composition) Total solution volume 25 μL Detection primer 10 pmol × 10 buffer 2.5 μL B-ddTTP 2000 pmol or B-ddATP 400 pmol Delta-Tth DNA polymerase 5 U PCR reaction solution (after treatment) 1 μL (ddNTP uptake reaction conditions) 92 1 minute 92 ° C 1 minute, 45 ° C 1 minute (20 cycles) 75 ° C 1 minute (5) Hybridization in a microtiter plate D in N NaOH
After denaturing NA, 20 μL of each sample was added to 100 μL of a solution of 200 mM citrate-phosphate buffer (pH 6.0), 2% SDS, 750 mM NaCl, and the mixture was added to the microtiter plate to which the capture probe of (2) above was bound. I put it in. Liquid paraffin was overlaid to prevent evaporation, and shaken at 37 ° C for 30 minutes. by this,
The detection primer is specifically captured on the microtiter plate by the immobilized capture probe.

Next, the solution was replaced with 2 × SSC (pH 7.0) and 1% SDS. Similarly, in order to prevent evaporation, liquid paraffin was overlaid at 37 ° C.
Shake for 20 minutes. Then, 50 mM of streptavidin (DKO D0396) labeled with alkaline phosphatase was added.
The solution was replaced with 100 μL of a solution diluted 2000-fold with a solution of Tris-HCl buffer (pH 7.5) and 1% BSA, and shaken at 37 ° C. for 15 minutes.
Thereby, when B-ddNTP is incorporated into the captured detection primer, alkaline phosphatase-labeled streptavidin specifically binds to biotin. 250 μL of 50 mM Tris-HCl buffer (pH 7.5), 0.025%
After washing three times with Tween20 solution, dioxetane compound (trade name: CSPD; TR) which is a luminescent substrate of alkaline phosphatase
After injection of 50 µL of OPIX and keeping the mixture at 37 ° C for 15 minutes, the amount of luminescence was measured with a photon counter (Hamamatsu Photonics) in a dark room. (6) Results of human ACE gene single nucleotide polymorphism measurement Table 1 shows the results of detection of the human ACE gene inserted gene polymorphism detected in (5) above. The numerical value is the amount of light emission (cps: count / se
cond). As shown in Table 1, in the reaction system incorporating B-ddTTP, a sample containing allyl of T such as TT and TA emits a large amount of light, whereas a sample of AA containing no allyl of T has 3 kcps. Less than the amount of light emission. On the other hand, in the reaction system for incorporating B-ddATP, a sample containing allyl of A such as TA and AA emits a large amount of light, whereas a sample of TT containing no allyl of A has a light emission of less than 3 kcps. Stay. 3kc as cutoff
If ps is set, both the TA sample is positive, the TT sample is positive only T, and the AA sample is positive only A, so that SNP can be determined. As a whole, the positive signal of B-ddATP is higher than that of B-ddTTP, and a difference in the incorporation efficiency can be considered depending on the type of ddNTP, but it does not interfere with the judgment. The reason that the signal of phosphatase is higher than that of QIAquick as pretreatment is considered to be due to less loss of nucleic acid during pretreatment. In addition, the positive signal was clearly improved in the asymmetric PCR, confirming the effect of improving the uptake of B-ddNTP.

[0028]

[Table 1]

Embodiment 2 FIG. Detection of single nucleotide polymorphism in human CD14 receptor gene by combining PCR with the present invention Detection of SNP (CD14R-260 C / T) present at the 260th base upstream of the transcription start site of human CD14 receptor gene using the present invention I do.

In Example 2, the two types of primers, the CD14R-260SNP detection forward primer (SEQ ID NO: 7) and the CD14R-260SNP detection reverse primer (SEQ ID NO: 8) were used. This is to show. (1) Preparation of Necessary Oligonucleotides Oligonucleotide having sequence shown in SEQ ID NO: 5 (forward primer for amplifying CD14R gene), oligonucleotide having sequence shown in SEQ ID NO: 6 (CD14R gene amplification) Reverse primer for use), oligonucleotide having the sequence shown in Sequence Listing / SEQ ID NO: 7 (forward primer for CD14R-260SNP detection), oligonucleotide having the sequence shown in Sequence Listing / SEQ ID NO: 8 (reverse primer for detecting CD14R-260SNP) An oligonucleotide having the sequence shown in SEQ ID NO: 9 (oligonucleotide for dT20 capture probe) was synthesized. The oligonucleotide for dT20 capture probe has an amino linker arm at the 5 'end. (2) Binding of oligonucleotide for capture probe to microtiter plate The oligonucleotide for capture probe synthesized in (1) above was bound to the inner surface of the microtiter plate via its linker arm. Linker oligonucleotide is 50mM borate buffer (pH10) 100mM
Dilute to a concentration of 0.05 pmol / μL in a MgCl ₂ solution, dispense 100 μL each into a microtiter plate (MicroFLUOR B, Dynatech), and leave at room temperature for about 15 hours to convert the linker oligonucleotide into a microtiter plate. Bond to the inner surface of the plate. After that, 0.1pmol dNTP
Replace with 0.5% PVP (polyvinyl pyrrolidone) 5 × SSC,
Blocking for suppressing non-specific reaction was performed at room temperature for about 2 hours. Finally, it was washed with 1 × SSC and dried. (3) Amplification by PCR of a region containing a single nucleotide polymorphism to be specified Oligonucleotide (forward primer for amplifying CD14R gene) having the sequence shown in SEQ ID NO: 5, sequence shown in SEQ ID NO: 6 Nucleic Acid Extraction Kit “QIAamp” (Qiagen) using oligonucleotides with reverse primers (CD14R gene amplification reverse primer)
A part of the CD14R gene was amplified by PCR from a human genomic DNA sample extracted from blood in step (1). In these DNA samples, the bases of CD14R-260 have been specified in advance by an ordinary sequencing method, and are three types of CC / CT / TT, respectively. PCR
Using KOD DNA polymerase (Toyobo), 35 cycles of amplification reaction were carried out under the conditions specified by the manufacturer. However,
Since the purpose of this PCR is simply to amplify a specific region of a gene, there is no particular limitation on the method of PCR and the type of enzyme. In addition, the reverse primer was injected 10 times the standard method during PCR, and the antisense strand was preferentially amplified.
(= Described in Table 2 as PCR "R increase") During the sample and PCR, forward primer was added 10 times of the standard method to amplify the sense strand preferentially (= described as PCR "F increase" in Table 2) )
There are also samples. Thereafter, the cells were treated with bovine small intestine alkaline phosphatase (Toyobo) at 37 ° C. for 15 minutes and then at 80 ° C. for 15 minutes. (PCR composition (Regular method)) Total solution volume 25 μL Forward primer 5 pmol (50 pmol) Reverse primer 5 pmol (50 pmol) × 10 buffer 2.5 μL 2 mM dNTP 2.5 μL KOD DNA polymerase 1U extraction DNA solution 100 ng (PCR conditions) 94 ° C., 5 minutes 94 ° C for 1 minute, 55 ° C for 2 minutes, 68 ° C for 1 minute (35 cycles) 68 ° C for 2 minutes (4) Specific ddNTP incorporation reaction The PCR product prepared in (3) was added with an SNP detection primer, specific
Add biotinylated ddNTP (B-ddNTP), DNA polymerase, etc.
Allow ddNTP to be taken up. This time, the types of SNPs are C and T, which are the bases of the sense strand and G and A, respectively, in the antisense strand. Therefore, CD
When using the 14R-260SNP detection forward primer (=
Primer "F" in Table 2) is B-ddCTP or B-ddTTP,
When using the reverse primer for CD14R-260SNP detection (=
B-ddGTP or B-ddATP was used for primer "R" in Table 2). In addition, a modified Tth DNA polymerase “Delta-Tth
DNA polymerase "(Toyobo). Since B-ddTTP and B-CTP have lower incorporation efficiencies than B-ddATP and B-ddGTP, they are frequently used. (Composition of ddNTP incorporation) Total solution volume 25μL Detection primer 10pmol × 10 buffer 2.5μL B-ddCTP 2000pmol or B-ddTTP 2000pmol or B-ddGTP 400pmol or B-ddATP 400
pmol Delta-Tth DNA polymerase 5U PCR reaction solution (after treatment) 1 μL (ddNTP incorporation reaction conditions) 92 ° C for 1 minute 92 ° C for 1 minute, 45 ° C for 1 minute (20 cycles) 75 ° C for 1 minute (5) Micro Hybridization in titer plate (4) Dilute the incorporation reaction solution 10-fold and add D in 0.3N NaOH.
Denature the NA and add 20 μL of each sample to a solution of 200 mM citrate-phosphate buffer (pH 6.0), 2% SDS, 750 mM NaCl 100
In addition to μL, the solution was placed in a microtiter plate to which the capture probe of the above (2) was bound. Liquid paraffin was overlaid to prevent evaporation, and shaken at 37 ° C for 30 minutes. Thereby, the detection primer is specifically captured on the microtiter plate by the immobilized capture probe.

Next, the mixture was replaced with 2 × SSC (pH 7.0) and 1% SDS. Similarly, in order to prevent evaporation, liquid paraffin was overlaid at 37 ° C.
Shake for 20 minutes. Then, 50 mM of streptavidin (DKO D0396) labeled with alkaline phosphatase was added.
The solution was replaced with 100 μL of a solution diluted 2000-fold with a solution of Tris-HCl buffer (pH 7.5) and 1% BSA, and shaken at 37 ° C. for 15 minutes.
Thereby, when B-ddNTP is incorporated into the captured detection primer, alkaline phosphatase-labeled streptavidin specifically binds to biotin. 250 μL of 50 mM Tris-HCl buffer (pH 7.5), 0.025%
After washing three times with Tween20 solution, dioxetane compound (trade name: CSPD; TR) which is a luminescent substrate of alkaline phosphatase
After injection of 50 µL of OPIX and keeping the mixture at 37 ° C for 15 minutes, the amount of luminescence was measured with a photon counter (Hamamatsu Photonics) in a dark room. (6) Results of measurement of human CD14 receptor gene single nucleotide polymorphism Table 2 shows the results of detecting the inserted gene polymorphism of the CD14 receptor gene detected in (5) above. Numerical values are luminescence (cps: co
unt / second). CC and GG, CT and GA, T in Table 2
T and AA are the same sample. As shown in Table 2, CD14R
When using -260SNP detection forward primer, B-ddC
In the reaction system incorporating TP, samples containing allyls of C, such as CC and CT, emit a high amount of luminescence, whereas C
In the TT sample containing no allyl, the luminescence is less than 3 kcps. On the other hand, in the reaction system in which B-ddTTP is incorporated, the amount of luminescence is high in a sample containing allyl of T such as TT and CT, whereas the amount of luminescence is less than 3 kcps in a sample of CC not containing allyl of T. Stay. CD14R-260SN
When a P-detection reverse primer is used, in a reaction system that incorporates B-ddGTP, a sample containing an allele of A, such as GG and GA, emits a large amount of light, whereas a sample of AA that does not contain an allyl of G is used. The light emission is less than 3kcps. On the other hand, in the reaction system that incorporates B-ddATP, a sample containing A allyl such as AA and GA emits a large amount of light, whereas a GG sample not containing allyl A has a light emission of less than 3 kcps. Stay. 3kc as cutoff
If ps is set, the SNP can be determined as in the case of the first embodiment. The results showed that it was possible to add dA 20mer to the 5 'end of the detection primer and detect with a dT 20mer capture probe. This makes it possible to use a common capture probe. Also asymmetric PC
The effect of R could be confirmed again.

[0032]

[Table 2]

[0033]

According to the present invention, there can be provided a nucleic acid sequence analysis method capable of easily detecting the type of nucleotide polymorphism in a specific nucleic acid sequence containing a single nucleotide polymorphism contained in a sample.

[0034]

[Sequence List] SEQUENCE LISTING <110> TOYO BOSEKI KABUSHIKI KAISHA <120> A METHOD FOR DETECTING SNPs <130> 20D00JP <140> <141> <160> 9 <170> PatentIn Ver. 2.1 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 1 TCGGGCTGGG AAGATCGAGC 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: primer <400> 2 CTCTGCCCCT TCTCCTGCGC 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 CTGCCGGGTC CCCATCTTC 19 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 GAAGATGGGG ACCCGGCAG 19 <210> 5 <211> 20 <212> DNA <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 CGCCTGAGTC ATCAGGACAC 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 CCTTTCCTGG AAATATTGCA 20 < 210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 AAAAAAAAAA AAAAAAAAAA CAGAATCCTT CCTGTTACGG 40 <210> 8 <211> 40 <212> DNA < 213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 8 AAAAAAAAAA AAAAAAAAAAAAGAGATGTTTC AGGGAGGGGG 40 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 9 TTTTTTTTTT TTTTTTTTTT 20

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Toshiya Aono 2-1-1 Katata, Otsu-shi, Shiga Prefecture Inside Toyobo Co., Ltd. Research Institute (72) Inventor Satoko Yoshiga 2-1-1 Katata, Otsu-shi, Shiga Prefecture 4B024 AA11 AA20 CA01 CA09 HA12 HA19 4B063 QA12 QA18 QQ42 QR08 QR32 QR56 QR62 QS03 QS34 QX02

Claims (8)

[Claims] 1. A method for detecting a single nucleotide polymorphism on the sequence of a chromosome or a fragment thereof, comprising: a nucleic acid primer having a base adjacent to the polymorphic base at the 3 ′ end; A sample nucleic acid is reacted with a reaction solution containing any one type of dideoxynucleotide or a mixture thereof, a DNA polymerase and a composition necessary for expressing its activity, and detects the presence of dideoxynucleotide incorporation into a nucleic acid primer. A method for detecting the type of base to be identified by the method. 2. The method of claim 1, wherein the distinguishable feature of the incorporated dideoxynucleotide is a biotin group. 3. The method according to claim 1, wherein after the incorporation reaction, the nucleic acid primer is captured and detected by hybridizing with the capture probe. 4. The method according to claim 1, wherein the DNA polymerase is heat-resistant, and the sensitivity is improved by repeating nucleic acid denaturation and annealing / incorporation by a temperature cycle. 5. The method according to claim 1, wherein the ratio of nucleic acid primers is made non-uniform so that the strand on the side to be detected is preferentially amplified, and the sample nucleic acid used for detection is amplified in advance. The method described in. 6. The method according to claim 5, wherein an amplification primer that hybridizes to the same sample nucleic acid strand as the nucleic acid primer is increased by 5 to 10 times as compared with an amplification primer that hybridizes to the opposite sample nucleic acid strand. The described method. 7. The method according to claim 1, wherein after amplifying a sample nucleic acid used for detection in advance, dephosphorylation of various deoxynucleotides, which are obstacles in detecting a single nucleotide polymorphism, with a phosphatase. The described method. 8. An oligonucleotide having a common sequence arbitrarily set in advance is bound to the 5 'end of each nucleic acid primer, and the nucleic acid primer is captured using a capture probe complementary to the oligonucleotide having the common sequence. ,
The method of claim 1, wherein detecting.